Prieto Daniel, Aparicio Gonzalo, Machado Matías, Zolessi Flavio R
Institut Pasteur de Montevideo.
Institut Pasteur de Montevideo; Sección Biología Celular, Facultad de Ciencias, Universidad de la República.
J Vis Exp. 2015 May 2(99):e52769. doi: 10.3791/52769.
Methyl green has long been known as a histological stain with a specific affinity for DNA, although its fluorescent properties have remained unexplored until recently. In this article, we illustrate the method for preparing a methyl green aqueous stock solution, that when diluted can be used as a very convenient fluorescent nuclear label for fixed cells and tissues. Easy procedures to label whole zebrafish and chick embryos are detailed, and examples of images obtained shown. Methyl green is maximally excited by red light, at 633 nm, and emits with a relatively sharp spectrum that peaks at 677 nm. It is very inexpensive, non-toxic, highly stable in solution and very resistant to photobleaching when bound to DNA. Its red emission allows for unaltered high resolution scanning confocal imaging of nuclei in thick specimens. Finally, this methyl green staining protocol is compatible with other cell staining procedures, such as antibody labeling, or actin filaments labeling with fluorophore-conjugated phalloidin.
长期以来，甲基绿一直被认为是一种对DNA具有特殊亲和力的组织学染色剂，不过其荧光特性直到最近才得到探索。在本文中，我们阐述了制备甲基绿水溶液储备液的方法，该储备液稀释后可作为一种非常便捷的荧光核标记物用于固定细胞和组织。文中详细介绍了标记整个斑马鱼和鸡胚胎的简便程序，并展示了所获得图像的示例。甲基绿在633 nm的红光激发下达到最大激发效果，并以相对较窄的光谱发射，峰值在677 nm。它非常便宜、无毒、在溶液中高度稳定，并且与DNA结合时非常抗光漂白。其红色发射允许对厚标本中的细胞核进行未经改变的高分辨率扫描共聚焦成像。最后，这种甲基绿染色方案与其他细胞染色程序兼容，例如抗体标记或用荧光团偶联的鬼笔环肽标记肌动蛋白丝。
